An impedance labeling free electrochemical aptamer sensor based on tetrahedral DNA nanostructures for doxorubicin determination.

Based on the electrochemical impedance method, a marker-free biosensor with aptamer as a biometric element was developed for the determination of doxorubicin (DOX). By combining aptamer with rigid tetrahedral DNA nanostructures (TDNs) and fixing them on the surface of gold electrode (GE) as biometric elements, the density and directivity of surface nanoprobes improved, and DOX was captured with high sensitivity and specificity. DOX was captured by immobilized aptamers on the GE, which inhibited electron transfer between the GE and [Fe(CN)6]3-/4- in solution, resulting in a change in electrochemical impedance. When the DOX concentration was between 10.0 and 100.0 nM, the aptasensor showed a linear relationship with charge transfer resistance, the relative standard deviation (RSD) ranged from 3.6 to 5.9%, and the detection limit (LOD) was 3.0 nM. This technique offered a successful performance for the determination of the target analyte in serum samples with recovery in the range 97.0 to 99.6% and RSD ranged from 4.8 to 6.5%. This method displayed the advantages of fast response speed, good selectivity, and simple sensor structure and showed potential application in therapeutic drug monitoring.

General Synthesis of N-CF3 Heteroaryl Amides via Successive Fluorination and Acylation of Sterically Hindered Isothiocyanates.

We report the one-pot synthesis of N-CF3 heteroaryl amides (NTFMHA) from heteroaryl carboxylic acids and sterically hindered isothiocyanates, including various amino acid analogues, in the presence of AgF. The key to this reaction is the utilization of free heteroaryl acyl chlorides, rather than their corresponding hydrochloride salts. This method represents a complementary method of our previous work and enables modification to a variety of previously inaccessible structures, including α-tertiary amines and N-CF3-modified pharmaceuticals.

Fluorescence Biosensor for One-Step Simultaneous Detection of Mycobacterium tuberculosis Multidrug-Resistant Genes Using nanoCoTPyP and Double Quantum Dots.

The diagnosis of multidrug-resistant tuberculosis (MDR-TB) is crucial for the subsequent drug guidance to improve therapy and control the spread of this infectious disease. Herein, we developed a novel florescence biosensor for simultaneous detection of Mycobacterium tuberculosis (Mtb) multidrug-resistant genes (rpoB531 for rifampicin and katG315 for isoniazid) by using our synthesized nanocobalt 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (nanoCoTPyP) and double quantum dots (QDs). Several nanoCoTPyPs with different charges and morphology were successfully prepared via the surfactant-assisted method and their quenching ability and restoring efficiency for DNA detection were systematically analyzed. It was found that spherical nanoCoTPyP with positive charge exhibited excellent quenching effect and sensing performance for the two DNAs' detection due to its affinity differences towards single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). ssDNA attached on QDs (QDs-ssDNA) was specifically hybridized with targets to form QDs-dsDNA, resulting in fluorescence recovery due to the disruption of the interactions between nanoCoTPyP and ssDNA. Two drug-resistant genes could be simultaneously quantified in a single run and relatively low limits of detection (LODs) were obtained (24 pM for T1 and 20 pM for T2). Furthermore, the accuracy and reliability of our method were verified by testing clinical samples. This simple and low-cost approach had great potential to be applied in clinical diagnosis of MDR-TB.

Coulometric back titration based on all-solid-state electrodes for phenylephrine hydrochloride determination.

Existing coulometric titration generally used liquid reference electrodes, with limitations of frequent maintenance and liquid leakage risk and requiring vertical working position. Herein, we proposed an all-solid coulometric titration based on the glassy carbon electrode (GCE) and preliminarily utilized it in phenylephrine hydrochloride (PHE) analysis with a universal back titration program. Subsequently, the long-term stability of the GCE in bromine and iodine solution was evaluated comprehensively by electrochemical characterization and titration measurement. The results presented an excellent linear relationship between electrolysis time and PHE concentration from 0.3 to 20 mM, with a limit of detection of 0.07 mM (3 S0/S). It also showed an excellent standard recovery rate and a short testing time within 10 min. Compared with bromometry in the pharmacopoeia, the all-solid coulometric titration exhibited higher accuracy and precision in formulation detection with simpler operation. The long-term stability experiments indicated that the potential difference mutation was unaffected by the electrochemical property of the GCE. Conclusively, this enabling work provided an all-solid coulometric titration for drug determination without maintenance in the long term and contributed to its further integration and industrialization.

Determination of tuberculosis-related volatile organic biomarker methyl nicotinate in vapor using fluorescent assay based on quantum dots and cobalt-containing porphyrin nanosheets.

Methyl nicotinate (MN) is a representative and typical volatile organic marker of Mycobacterium tuberculosis, and the specific detection of MN in human breath facilitates non-invasive, rapid, and accurate epidemic screening of tuberculosis infection. Herein, we constructed a fluorescent assay consisted of CdTe quantum dots (QD) and cobalt-metalized tetrakis(4-carboxyphenyl) porphyrin (CoTCPP) nanosheets to determine methyl nicotinate (MN) in vapor samples. Red-emission QD (λex=370 nm, λem=658 nm) acts as signal switches whose fluorescence signals can be effectively quenched by CoTCPP nanosheets but restored in the presence of MN. The strategy relied on the distinct binding affinity of cobalt ion and MN. MN restored the fluorescence of QD quenched by CoTCPP in a concentration-dependent manner, which exhibited a well-linear relationship in the range 1-100 µM, and a limit of detection of 0.59 µM. The proposed platform showed sensitivity and selectivity to detect MN in vapor samples with satisfactory RSD below 3.33%. The method is cheap, simple, and relatively rapid (detected within 4 min), which suggests a potential in tuberculosis diagnosis in resource- and professional-lacked areas.

An integrated microfluidics for assessing the anti-aging effect of caffeic acid phenethylester in Caenorhabditis elegans.

Aging is a fundamental and fascinating process. Anti-aging research tried to find the mysteries about the human lifespan. To investigate the longevity-extending role of caffeic acid phenethylester (CAPE) and reveal the possible regulation mechanism, the long-term cultivation under well-defined environments, real-time monitoring, and live imaging is highly desired. In this paper, a well-designed microfluidic device was proposed to analyze the anti-aging effect of CAPE in Caenorhabditis elegans. With the combined use of multiple functional units, including micro-pillar, worm responder, a branching network of distribution channels, and microchambers, the longitudinal measurements of the exact number of worms throughout the whole lifespans is possible. Meanwhile, the brief cooling function of temperature-controllable system can achieve temporary and repeated immobilization of nematodes for fluorescence imaging. Our research data showed that CAPE can increase the survival of worms under normal and stress condition, including heat stress and paraquat-induced oxidative stress. The further studies revealed the anti-aging mechanism of CAPE. This proposed strategy and device would be a useful platform to facilitate future anti-aging studies and the finding of new lead compounds.

Sensitive analysis of doxorubicin and curcumin by micellar electromagnetic chromatography with a double wavelength excitation source.

Doxorubicin has been extensively used to treat cancers, and there are recent findings that the anticancer activities can be enhanced by curcumin. Although the two compounds have native fluorescence, they can hardly be quantified directly simultaneously using the laser-induced fluorescence (LIF) detection method. To avoid complex fluorescence derivatization and introduction of interfering components, a highly sensitive double wavelength excitation source LIF (D-W-Ex-LIF) detector composed of a 445-nm and 488-nm commercial laser diode was constructed to detect them simultaneously. Rhodamine 6G was selected as an internal standard, because its fluorescence can be excited at 445 nm and 488 nm. The native fluorescence of doxorubicin and curcumin and their resolution were enhanced by introducing mixed micelles. The optimal electrophoretic separation buffer was 10 mM borate buffer containing 20 mM Triton X-100, 5 mM sodium dodecyl sulfate, and 30% (v/v) methanol at pH 9.00. Therefore, the developed method was specific, accurate, and easily operable. Its limits of detection for doxorubicin and curcumin in human urine samples were 4.00 × 10-3 and 1.00 × 10-2 µg/mL, respectively, and the limits of quantification were 1.00 × 10-2 and 3.00 × 10-2 µg/mL, respectively. The recoveries were 94.9-109.1%. Graphical abstract.

DNA nanotetrahedron linked dual-aptamer based voltammetric aptasensor for cardiac troponin I using a magnetic metal-organic framework as a label.

An ultrasensitive voltammetric aptasensor was constructed to analyze cardiac troponin I (cTnI). It is based on DNA nanotetrahedron (NTH) linked dual-aptamer (dAPT) and magnetic metal organic frameworks (mMOFs) of type Fe3O4@UiO-66. Firstly, the DNA NTH linked dAPT (Tro4 and Tro6) were immobilized on a gold electrode for improving the capture efficiency of cTnI. The novel mMOFs Fe3O4@UiO-66 was then decorated by Au@Pt nanoparticles (Au@PtNPs), horseradish peroxidase (HRP), G-quadruplex/hemin (GQH) DNAzyme, and two types of aptamers to form signaling nanoprobes. In the presence of cTnI, an aptamer-protein-nanoprobe sandwich-type structure is formed. Afterward, the nanoprobes including enzyme, GQH DNAzyme and Fe3O4@UiO-66/Au@PtNP were utilized to catalyze the oxidation of hydroquinone by hydrogen peroxide for the electrochemical signals amplification, typically at a working potential of -0.1 V (vs. Ag/AgCl). The voltammetric signal increases linearly in the 0.01 to 100 ng·mL-1 cTnI concentration range, and the detection limit is 5.7 pg·mL-1. Graphical abstract An ultrasensitive voltammetric aptasensor was constructed to analyze cardiac troponin I (cTnI) based on DNA nanotetrahedron linked dual-aptamer and magnetic metal organic frameworks of type Fe3O4@UiO-66. The results indicated the aptasensor has a wide linear response range (0.01 to 100 ng/mL) and low detection limit (5.74 pg/mL) for cTnI. GE: gold electrode; MCH: 6-Mmercapto-1-hexanol; HRP: horseradish peroxidase; HQ: hydroquinone; BQ: benzoquinone.

Dual-aptamer-based voltammetric biosensor for the Mycobacterium tuberculosis antigen MPT64 by using a gold electrode modified with a peroxidase loaded composite consisting of gold nanoparticles and a Zr(IV)/terephthalate metal-organic framework.

An ultrasensitive aptasensor is described for the voltammetric determination of the Mycobacterium tuberculosis antigen MPT64 in human serum. Firstly, an amino-modified Zr(IV) based metal-organic framework (MOF; type UiO-66-NH2; made up from Zr6O32 units and 2-amino-terephthalate linkers) with a high specific surface was synthesized and used as the carrier of the gold nanoparticles and the aptamers. Then the signalling nanoprobe was fabricated after the horseradish peroxidase was cast on the nanomaterials. The two aptamers with synergistic effect on binding MPT64 were anchored on the gold electrode. Differential pulse voltammetry indicated that the peak current is highest if the ratio of the two aptamers is 1:1. The assay has a wide linear response range (0.02 to 1000 pg·mL-1 of MPT64) and a 10 fg·mL-1 detection limit at a working potential of around -96 mV (vs Ag/AgCl). The results show this biosensor to be a viable tool for detection of tuberculosis at an early stage. Graphical abstract Schematic presentation of the construction of the nanoprobe and biosensor. Firstly, the surface of UiO-66-NH2 was anchored to gold nanoparticles (AuNPs). A dual-aptamer and HRP were added to form the signalling nanoprobe (Aptamer/HRP/AuNPs/UiO-66-NH2). Then, the aptamers I and II were attached on the surface of gold electrode and 6-mercapto-1-hexanol was used to block the uncovered active site of the gold electrode. Finally, after incubation with MPT64, the signalling nanoprobe was dropped on the modified electrode and the DPV measurements was used for the analysis of Mycobacterium tuberculosis antigen MPT64. (PVP: poly(vinyl pyrrolidone); HRP: horseradish peroxidase; MCH: 6-Mercapto-1-hexanol; HQ: hydroquinone; BQ: benzoquinone).

The protection of novel 2-arylethenylquinoline derivatives against impairment of associative learning memory induced by neural Aß in C. elegans Alzheimer's disease model.

Cerebral deposition of amyloid ß-peptide (Aß), a fundamental feature of Alzheimer's disease (AD), damages the neurocytes and impairs the cognition functions and associative learning memory of AD patients. A series of novel 2-arylethenylquinoline derivatives were synthesized and evaluated in our previous study, which inhibited Aß aggregation in vitro effectively at the concentration of 20 µmol/L and exhibited high antioxidant activity. In order to verify the capacity of anti-AD in vivo, the transgenic Caenorhabditis elegans (C. elegans) strain CL2355 expressing neural Aß was employed as the AD model to investigate the neuroprotective activity of seven high-potential compounds (4a1, 4a2, 4b1, 4b2, 4c1, 4c2, 4c3) selected from those derivatives. Learning memory associated chemotaxis assay was performed to evaluate the neural repairment capacity. The underlying mechanism was investigated by mRNA analysis of Aß gene and heat shock protein genes (hsp-16.1 and hsp-16.2) and Western blot of Aß. Our data indicated that among seven tested compound, 4b1 and 4c2 reduced Aß-induced stress, suppressed the expression of neural Aß monomers and toxic oligomers, and recovered the damaged associative learning memory in C. elegans AD model. These findings further confirmed their potentials to become valuable agents for AD therapy.

A simple and compact fluorescence detection system for capillary electrophoresis and its application to food analysis.

A novel fluorescence detection system for CE was described and evaluated. Two miniature laser pointers were used as the excitation source. A Y-style optical fiber was used to transmit the excitation light and a four-branch optical fiber was used to collect the fluorescence. The optical fiber and optical filter were imported into a photomultiplier tube without any extra fixing device. A simplified PDMS detection cell was designed with guide channels through which the optical fibers were easily aligned to the detection window of separation capillary. According to different requirements, laser pointers and different filters were selected by simple switching and replacement. The fluorescence from four different directions was collected at the same detecting point. Thus, the sensitivity was enhanced without peak broadening. The fluorescence detection system was simple, compact, low-cost, and highly sensitive, with its functionality demonstrated by the separation and determination of red dyes and fluorescent whitening agents. The detection limit of rhodamine 6G was 7.7 nM (S/N = 3). The system was further applied to determine illegal food dyes. The CE system is potentially eligible for food safety analysis.

Inhibitory effect of Aristolochia fruit on Cytochrome P450 isozymes in vitro and in vivo.

The mature fruits of Aristolochia debilis, known in China by the name, "Madouling" has been popularly prescribed in Asia, particularly in China, to treat a range of conditions including gynaecological problems, arthritis and wound healing. This study was aimed to evaluate the potential effect of Madouling on the cytochrome P450 (CYP) isozymes in vitro in microsomal fractions and in vivo in rats. The influence of Madouling on CYPs activity was first explored by an in vitro method of estimating levels of four respective metabolites in rat liver microsomes. The results were re-examined in vivo in rats by using a cocktail approach involving the probe drugs theophylline, tolbutamide, chlorzoxazone and dapsone. Pharmacokinetics of the four substrates was used to analyze the activities of the targeting isozymes. In vitro study revealed that Madouling decreased the activity of CYP1A2, 3A1 and 2E1. However, no significant influence on CYP2C6 was found. These results coincided with those of in vivo study to a great degree except that in vivo estimation the herb didn't inhibit CYP1A2 significantly. From the data obtained, Madouling is suggested as a candidate for clinically significant CYP interactions. Drug co-administrated with Madouling may need dose adjustment.

Ultrasensitive electrochemical detection of microRNA based on an arched probe mediated isothermal exponential amplification.

In this work, a simple and label-free electrochemical biosensor is developed for microRNA (miRNA) detection on the basis of an arched probe mediated isothermal exponential amplification reaction (EXPAR). The arched probe assembled on the electrode surface consists of two strands that are partially complementary to each other at both ends. The target can hybridize with the complementary sequence of the arched structure, leading to the cleavage of the probe. The strand fixed on the surface of the electrode self-assembles, in the presence of hemin, to G-quadruplex unit, yielding electrochemical signals. The other strand liberated into the solution triggers the EXPAR to recycle and regenerate targets. This method exhibits ultrahigh sensitivity toward miRNA with detection limits of 5.36 fM and a detection range of 3 orders of magnitude. The biosensor is capable of discriminating a single-nucleotide difference between concomitant miRNA and performs well in analyzing crude extractions from cancer cell lines.

Molecularly imprinted solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of safranine T in wolfberry.

A method was developed to sensitively determine safranine T in wolfberry by molecularly imprinted solid-phase extraction (MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using safranine T, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions and the morphologies of inner polymers were characterized by scanning electron microscopy (SEM). The mean recoveries of safranine T in wolfberry ranged from 91.2 % to 92.9 % and the intraday and interday relative standard deviation (RSD) values all ranged from 3.4 % to 4.2 %. Good linearity was obtained over 0.001-1.0 µg mL(-1) (r = 0.9999) with a detection limit (S/N = 3) of 0.4 ng g(-1). Under the selected conditions, enrichment factors of over 90-fold were obtained and the extraction on the monolithic column effectively cleaned up the wolfberry matrix. The results demonstrated that the proposed MISPE-HPLC-LIF method could be applied to sensitively determine safranine T in wolfberry.

A two-electrode system-based electrochemiluminescence detection for microfluidic capillary electrophoresis and its application in pharmaceutical analysis.

A two-electrode configuration powered by batteries was designed for a microchip capillary electrophoresis-electrochemiluminescence system. A home-made working electrode for end-column mode detection and wall-jet configuration was made up of a platinum wire (0.3 mm diameter) and a quartz capillary (320 µm internal diameter). The platinum wire served as a pseudoreference electrode. The configuration of the detection power supply comprised two D-size batteries (connected in series), a switch, and an adjustable resistor. The microchip consisted of two layers: the bottom layer was a glass sheet containing injection and separation channels; the upper layer was polydimethylsiloxane block. In order to reduce the loss of electrochemiluminescence signal, a coverslip (0.17 mm thickness) was used as the floor of the detection reservoir. The performance of the system was demonstrated by separation and detection of atropine, anisodamine and proline. The linear response for proline ranged from 5 µM to 100 µM (r = 0.9968), and the limit of detection was 1.0 µM (S/N = 3). The system was further applied to the measurement of atropine in atropine sulfate injection solutions with the limit of detection 2.3 µM. This new system is a potential tool in pharmaceutical analysis.

Enhanced Electrochemical Characterization of the Immune Checkpoint Protein PD-L1 using Aptamer-Functionalized Magnetic Metal-Organic Frameworks.

Programmed death ligand 1 (PD-L1) is highly expressed in cancer cells and participates in the immune escape process of tumor cells. However, as one of the most promising biomarkers for cancer immunotherapy monitoring, the key problem ahead of practical usage is how to effectively improve the detection sensitivity of PD-L1. Herein, an electrochemical aptasensor for the evaluation of tumor immunotherapy is developed based on the immune checkpoint protein PD-L1. The fundamental principle of this method involves the utilization of DNA nanotetrahedron (NTH)-based capture probes and aptamer-modified magnetic metal-organic framework nanocomposites as signaling probes. A synergistic enhancement is observed in the electrocatalytic effect between Fe3O4 and UiO-66 porous shells in Fe3O4@UiO-66 nanocomposites. Therefore, the integration of aptamer-modified Fe3O4@UiO-66@Au with NTH-assisted target immobilization as an electrochemical sensing platform can significantly enhance sensitivity and specificity for target detection. This method enables the detection of targets at concentrations as low as 7.76 pg mL-1 over a wide linear range (0.01 to 1000 ng mL-1). The authors have successfully employed this sensor for in situ characterization of PD-L1 on the cell surface and for monitoring changes in PD-L1 expression during drug therapy, providing a cost-effective yet robust alternative to highly expensive and expertise-dependent flow cytometry.

Research progress of metal-organic framework nanozymes in bacterial sensing, detection, and treatment.

The high efficiency and specificity of enzymes make them play an important role in life activities, but the high cost, low stability and high sensitivity of natural enzymes severely restrict their application. In recent years, nanozymes have become convincing alternatives to natural enzymes, finding utility across diverse domains, including biosensing, antibacterial interventions, cancer treatment, and environmental preservation. Nanozymes are characterized by their remarkable attributes, encompassing high stability, cost-effectiveness and robust catalytic activity. Within the contemporary scientific landscape, metal-organic frameworks (MOFs) have garnered considerable attention, primarily due to their versatile applications, spanning catalysis. Notably, MOFs serve as scaffolds for the development of nanozymes, particularly in the context of bacterial detection and treatment. This paper presents a comprehensive review of recent literature pertaining to MOFs and their pivotal role in bacterial detection and treatment. We explored the limitations and prospects for the development of MOF-based nanozymes as a platform for bacterial detection and therapy, and anticipate their great potential and broader clinical applications in addressing medical challenges.

Quantification of lactate in synovia by microchip with contactless conductivity detection.

This article describes the determination of lactate in synovia by microchip capillary electrophoresis (MCE) integrated with contactless conductivity detection (CCD). The optimal running buffer consists of 10mM tris(hydroxymethyl)aminomethane, 1 mM HCl, and 0.1 mM hexadecyltrimethylammonium bromide (pH 9.1). The quantitative measurement of lactate in dilute synovia samples can be finished in less than 40s. The results indicated that the peak area had a good linear relationship with lactate concentration in the range of 20 to 1000 µM, and the correlation coefficient was 0.9984. The average recovery was from 96.6% to 106.1%, and the interday relative standard deviation was less than 4.0% (n=6). The limit of detection (signal/noise=3) reached 6.5 µM. To validate the assay results, we compared the current method with the high-performance liquid chromatography method by measuring lactate in synovia samples. The data analysis verified that there was no significant difference between the two methods. Due to significant features such as low cost, integration, and miniaturization, the MCE-CCD method may have great potential in clinical diagnosis.

A microfluidic device for rapid screening of chemotaxis-defective Caenorhabditis elegans mutants.

Behavioral Caenorhabditis elegans mutants are sought for the purposes of neurobiological research. Until now, large numbers of worms with neuronal defects have been obtained through mutagenesis techniques. However, the existing screening procedures are not only time-consuming and low-throughput, but also tedious and labor-intensive. Therefore, developing a rapid and convenient method to overcome these difficulties is necessary. The present study demonstrates for the first time a microdevice for the rapid screening of chemotaxis-defective mutants based on their chemotactic response. The microchip is capable of automatic introduction, local immobilization, and controllable generation of concentration gradients during the screening assays. With this device, six C. elegans behavioral assays can be performed using various attractants without requiring anesthetics for local capture, and ten mutants are effectively isolated from 10(4) mutagenized worms in 100 min. The microfluidics-based method is robust enough to sort the chemotaxis-defective worms with 91 % accuracy from a large population of wild type animals during a mutagenesis screen.

Development of a microchip-pulsed electrochemical method for rapid determination of L-DOPA and tyrosine in Mucuna pruriens.

L-3,4-dihydroxyphenylalanine (L-DOPA) is a well-recognized therapeutic compound to Parkinson's disease. Tyrosine is a precursor for the biosynthesis of L-DOPA, both of which are widely found in traditional medicinal material, Mucuna pruriens. In this paper, we described a validated novel analytical method based on microchip capillary electrophoresis with pulsed electrochemical detection for the simultaneous measurement of L-DOPA and tyrosine in M. pruriens. This protocol adopted end-channel amperometric detection using platinum disk electrode on a homemade glass/polydimethylsiloxane electrophoresis microchip. The background buffer consisted of 10 mM borate (pH 9.5) and 0.02 mM cetyltrimethylammonium bromide, which can produce an effective resolution for the two analytes. In the optimal condition, sufficient electrophoretic separation and sensitive detection for the target analytes can be realized within 60 s. Both tyrosine and L-DOPA yielded linear response in the concentration range of 5.0-400 µM (R(2) > 0.99), and the LOD were 0.79 and 1.1 µM, respectively. The accuracy and precision of the established method were favorable. The present method shows several merits such as facile apparatus, high speed, low cost and minimal pollution, and provides a means for the pharmacologically active ingredients assay in M. pruriens.